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@PHDTHESIS{Lehmann:975823,
author = {Lehmann, Sonja Maria},
othercontributors = {Spehr, Marc and Leube, Rudolf and Zimmer-Bensch, Geraldine
Marion},
title = {{M}echanisms and functional consequences of impaired
keratin network formation in genetic skin disorders;
[überarbeitete {A}uflage]},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2023-12172},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2022},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University 2023. - Überarbeitete Auflage mit Korrektur von
Abbildungen; Dissertation, RWTH Aachen University, 2022},
abstract = {One of the most important barriers of the body is the skin.
Its outermost stratified layer, the epidermis, is mainly
composed of differentiating keratinocytes. The integrity of
the epidermis is to a large extent provided by structural
elements such as the keratin intermediate filament
cytoskeleton. The importance of keratin networks is
emphasised by mutation-induced keratinopathies such as
Epidermolysis bullosa simplex (EBS) or Pachyonychia
congenita (PC), caused by mutations in keratins 5/14 and
keratins 6/16/17, respectively. EBS is characterised by
trauma-induced blister formation due to rupture of
keratinocytes in the basal epidermal layer, which is caused
by the disruption of the keratin filament network into
granular structures. PC patients, on the other hand, display
epidermal thickening with extreme hyperkeratosis in certain
skin areas, such as the foot soles. This work aimed to gain
further insight into the different pathogeneses, in
particular to apply novel image analysis tools to
quantitatively describe mutant EBS keratin granules, and to
investigate whether autophagy of mitochondria, representing
an essential step of epidermal differentiation, is disturbed
in PC. Using live-cell microscopy of epithelial cells stably
overexpressing fluorophore-tagged EBS-mutant keratins, an
automated tracking routine was established. It allowed a
detailed quantitative analysis of different parameters of
mutant keratin granule dynamics. In particular, it was shown
that the mutant granules are initially formed in the
outermost lamellum of epithelial cells. Subsequently, they
grow up to a plateau size, and are constitutively
transported inwards with a velocity of approximately 0.5
µm/min. Most keratin granules display multiple fusion
events with other granules during their lifetime before they
rapidly disassemble at the boundary of the lamellum and the
inner cytoplasm. Their transport highly resembles
actin-dependent transport, and pharmacological inhibition of
the actin motor protein non-muscle myosin II significantly
reduced their dynamics. Fluorescence recovery after
photobleaching experiments furthermore revealed that the
granules rapidly exchange soluble keratins with the
surrounding cytoplasm and within the granule itself. Thus,
the formation of EBS-related keratin granules is based on
liquid-liquid phase separation (LLPS). The kinase DYRK was
investigated next, based on its ability to dissolve
different LLPS condensates. Although a clear colocalisation
of different overexpressed DYRK isoforms and mutant keratin
granules was shown in patient-derived EBS keratinocytes,
pharmacological inhibition of the kinases did not alter the
percentage of granule-containing cells in keratinocyte cell
clones stably overexpressing fluorophore-tagged mutant
keratins. In the second part of this work, functional
consequences of mutant keratins on mitochondrial integrity
were examined. First, the mitochondrial age was determined
by a fluorescent reporter in both EBS and PC keratinocytes.
This revealed that PC cells contain increased amounts of
overaged mitochondria, which was not the case for EBS.
Furthermore, contact sites between mitochondria and the
endoplasmic reticulum are reduced in PC. The expression of
early mitophagy markers is not changed, but clearance of
mitochondria is severely impaired in PC keratinocytes.
Although they are able to form autolysosomes, these
structures were shown to accumulate in PC. Assessment of
lysosomal function revealed defective enzymatic capacity and
the mitochondrial overaging phenotype could be mimicked by
lysosomal pH modifications in healthy cells. Thus, it can be
concluded that the process of autolysosomal recycling, which
is essential for macroautophagy, is impaired in PC, which
results in impaired mitophagy. Overall, this work
established new image analysis tools which allowed a
detailed quantification of mutant keratin dynamics, and
elucidated that mutant keratins modulate mitophagy and
autolysosomal recycling in PC keratinocytes.},
cin = {163310 / 160000},
ddc = {570},
cid = {$I:(DE-82)163310_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2023-12172},
url = {https://publications.rwth-aachen.de/record/975823},
}
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